Induction of pluripotent status in somatic cells by directed reprogramming in-vitro, (induced pluripotent stem (iPS) cells) offers great potential for the generation of disease- and patient-specific cell lines and cell therapy. Particularly, iPS cells provide the basis for practical generation of patient-specific cells for customized transplantation. Generation of iPS cells from mouse and human cells starting from somatic fibroblasts has recently been reported.
Two independent groups have identified laboratory protocols to induce iPS cell reprogramming. Takahasi et al. have reported that mouse and human skin cells can be transformed into ES-like cells by transduction of four genes: OCT3/4, SOX2, KLF4, and c-MYC (Cell. (2007) 131:861-72). Subsequent report demonstrated generation without c-MYC, making the procedure less prone to side effects such as induction of malignancy in host animal models (see Nakagawa et al., Nat. Biotechnol. (2008) 26:101-6). A slightly different set of genes (OCT3, SOX2, NANOG and LIN28) has also been reported to reprogram human iPS cells. Such methods typically utilize selectable markers (e.g., neomycin resistance markers) to isolate iPS cells. However, alternative procedures that eliminate drug selection make such procedure more amenable to clinical applications in humans (Meissner et al. (2007) Nat. Biotechnol. 25: 1177-81.).
Although promising, iPS techniques have several shortcomings that limit application of this approach for use in the clinic, including: the potential of retroviruses to cause tumors in tissues derived from host iPS cells; low efficiency of induction (approximately 1 in 5000-10000 cells); the length of time the process requires (at least 20-24 days to generate and identify iPS cells); and the need to use drug resistance selection of iPS cells.
Thus, there remains an unmet need for patient-customized cell lines for cell therapy and tissue regeneration that are safe, can be rapidly prepared and identified in quantity without the use of antibiotics or other drug-based selection.
Ongoing basic and applied research in this field continues to elucidate important findings about the pluripotency status as well as means of induction of the “iPS” status. For instance, genome-wide analysis of two key histone modifications in iPS cells has indicated that iPS cells are highly similar to ES cells. In addition, it has been reported that transcription factor-induced reprogramming leads to the global reversion of the somatic epigenome into an ES-like state (Maheralli et al. (2007) Cell Stem Cell 1:55-70). iPS gene expression has been reported to be required for about 10 days, after which cells enter a self-sustaining pluripotent state suggesting that factor-induced reprogramming is a gradual process with defined intermediate cell populations that contain cells poised to become iPS cells (Stadtfeld et al. (2008) Cell Stem Cell. 2:230-40).
Two overlapping groups of pluripotency-associated transcription factors have been identified. The first group includes Nanog, Oct4, Sox2, Smad1 and Stat3. The second, smaller group includes c-Myc (an oncogene that boosts reprogramming efficiency), n-Myc, Zfx and E2f1. This may explain the characteristic cooperative function of pluripotency-promoting genes and the need to have a number of the key genes unregulated in iPS cells (Chen et. al. (2008) Cell 133: 1106-17). Finally, using a combined chemical and genetics approach for the generation of iPS cells, conditions that can potentially reduce the need for viral transduction of oncogenic transcription factors have been identified using neural progenitors and small molecules (Shi et al. (2008) Cell Stem Cell. 2:525-528.).
To date reprogrammed iPS cells have been generated from a variety of host cells including hematopoietic, hair follicular, dermal fibroblasts, neuronal cells, umbilical cord blood cells, adult ocular progenitor cells, and pancreatic islet progenitor cells. iPS cells have also been generated from host cells of a variety of animals including human, mouse, rat, monkey, cow, sheep, goat, pig, horse, dog, cat, rabbit, and chicken. Moreover, pluripotent stem cells such as iPS and hESC have been differentiated into a variety of cell types including heart muscle, liver, neuronal, hematopoietic, pancreatic, bone, skin, sperm and retinal pigment epithelial cells, and in at least one case, a complete animal has been generated with contributions from iPS cells.
Despite significant ongoing R&D efforts, the current unmet need for patient-customized cell lines that could be used for cell therapy and tissue regeneration is the main driver for development of alternative more practical iPS procedures, speeding the development of this early stage discovery phase procedure into the clinic.